She is a graduate student (MS) in the School of Aquatic and Fishery Sciences, at the University of Washington. We talk about her thesis work of developing a management strategy evaluation for Tanner crab in the Eastern Bering Sea, her job with the Natural Resources Consultants Incorporated, how she got into the crab world, and her plans after receiving her degree. She also shares some of her at-sea experience with surveying in the Bering Sea.

http://owl.fish.washington.edu/scaphapoda/grace/Crab-project/DecaPod/S1E13-Interview-Madi-Shipley.mp3
]]>https://bittercrab.wordpress.com/2018/11/21/decapod-s1e13-interview-with-madi-shipley/feed/0graceac9DecaPod S1E12: Crab Meeting #6https://bittercrab.wordpress.com/2018/11/08/decapod-s1e12-crab-meeting-6/
https://bittercrab.wordpress.com/2018/11/08/decapod-s1e12-crab-meeting-6/#respondThu, 08 Nov 2018 07:38:05 +0000http://bittercrab.wordpress.com/?p=273This week we talk about transcriptome assembly (with our new RNAseq data from NWGC!!) and next steps. Also, we decide that Qiagen RNeasy Kit works pretty well for extracting RNA, and will be used to create the remaining libraries for the project.
http://owl.fish.washington.edu/scaphapoda/grace/Crab-project/DecaPod/S1E12-Crab-Mtg-6.mp3
]]>https://bittercrab.wordpress.com/2018/11/08/decapod-s1e12-crab-meeting-6/feed/0graceac9BLAST with bad Trinity fasta, R plan for adding Qubit data, and Testing out RNeasy Plus Mirco Kit – Grace’s Lab Notebookhttps://bittercrab.wordpress.com/2018/11/01/blast-with-bad-trinity-fasta-r-plan-for-adding-qubit-data-and-testing-out-rneasy-plus-mirco-kit-graces-lab-notebook/
https://bittercrab.wordpress.com/2018/11/01/blast-with-bad-trinity-fasta-r-plan-for-adding-qubit-data-and-testing-out-rneasy-plus-mirco-kit-graces-lab-notebook/#respondThu, 01 Nov 2018 13:03:10 +0000http://bittercrab.wordpress.com/?p=264Continue reading BLAST with bad Trinity fasta, R plan for adding Qubit data, and Testing out RNeasy Plus Mirco Kit – Grace’s Lab Notebook]]>Today I ran BLAST with the bad fasta from the Trinity run from last weekend. Will look more at the notebook Steven sent me to do the BLAST stats, goslim, contigs, go slim tables, etc. I also have been getting some input from Sam as to how best to manage adding new Qubit data to a master file consisting of all the hemolymph sampling data joined with the Qubit data results. Finally, I tested out the RNeasy Plus Micro Kit on 4 samples from Day 26, and ran the Qubit.

The original RNA extraction method using RNAzol RT resulted in low RNA yields, as well as contaminated samples, likely due to excess salts. We have submitted a pooled sample to the NWGC for library prep and sequencing. The pooled sample consists of samples from day 26 (samples were taken in triplicate) from cold and ambient infected and uninfected treatments. This library will be used for gene discovery.

As we continue investigating better methods for RNA extraction, we will create libraries that will be used for comparison between treatment groups.

]]>https://bittercrab.wordpress.com/2018/09/05/graces-notebook-september-goals/feed/0genefishGrace’s Notebook: Speed Vac New Pool of 15 Sampleshttps://bittercrab.wordpress.com/2018/09/05/graces-notebook-speed-vac-new-pool-of-15-samples/
https://bittercrab.wordpress.com/2018/09/05/graces-notebook-speed-vac-new-pool-of-15-samples/#respondWed, 05 Sep 2018 20:42:06 +0000http://bittercrab.wordpress.com/?p=214Continue reading Grace’s Notebook: Speed Vac New Pool of 15 Samples]]>Today I speed vac-ed the new pool of 15 samples for a little over three hours. There is still more than 50ul in the tube, so I’ll put it back in the speed vac as soon as I am in tomorrow morning.

The pooled sample is 150ul (15 samples, each 10ul).
Sam and I went over to the Speed Vac in FSH. At 10:50am, it was started on medium heat.

At 2:10pm, there was still too much liquid. As soon as I am in tomorrow am, I’ll put it back in the speed vac.

Put the sample in (cap open) a slot that has a little bit of paper towel stuffed in the bottom. Close lid. Turn on the machine. Turn the heat to medium. After it runs for a bit, turn on the vacuum (turn yellow valve marked “Vac” toward the machine). Then open up the vacuum by turning the blue dial on top of the machine to “open” (to the right).

To open it later, close the vacuum (blue dial) and then wait til it stops spinning.

]]>https://bittercrab.wordpress.com/2018/09/05/graces-notebook-speed-vac-new-pool-of-15-samples/feed/0genefishGrace’s Notebook: Gave Pooled Sample to the NWGC for Library Prep and RNA-Seq!https://bittercrab.wordpress.com/2018/09/05/graces-notebook-gave-pooled-sample-to-the-nwgc-for-library-prep-and-rna-seq/
https://bittercrab.wordpress.com/2018/09/05/graces-notebook-gave-pooled-sample-to-the-nwgc-for-library-prep-and-rna-seq/#respondWed, 05 Sep 2018 20:41:57 +0000http://bittercrab.wordpress.com/?p=216Continue reading Grace’s Notebook: Gave Pooled Sample to the NWGC for Library Prep and RNA-Seq!]]>Today I finished speed vac-ing (medium heat) the pooled sample. It ended up being too low of volume (14.1ul), so I added 40.9ul of 0.1% DEPC-treated H20. I ran 2ul of the sample on Qubit (RNA HS), and got a reading of 20.4 ul (1,081 ng RNA in the sample)!!!! We FINALLY have a sample to send off for library prep and sequencing! After getting info from NWGC (Chris in the Nickerson Lab at Foege) I put the sample on dry ice and walked it over! It is now in their hands until we get the data back.

Sample prep

Yesterday wasn’t enough time on the speed vac. So I put it back on (on medium) at 10:10 am. I took it off at 1pm.

I checked the volume of the sample by sucking it all up in a pipet tip (set to 50ul). Then, I decreased the volume on the pipet tip until the liquid was at the tip. It showed that the volume was 14.1 ul. It needs to be at least 50ul. So I sucked the sample back up with the pipet set at 14.1 ul. It all fit.

Then I added 40.9ul of 0.1% DEPC-treated H20 so the final volume was 55ul and vortexed it for 10s.

I then used 2ul of the sample to run on the Qubit (RNA HS) to check the RNA quantity. It read as having 20.4ng/ul of RNA! Meaning that in the sample, we had ~ 1,081 ng of RNA!!! (NWGC requires a minimum of 1000ng of RNA in a sample at least 50ul in volume).

I contacted NWGC to get info on what all they needed before I walked the sample over.

I brought it over on dry ice and handed it to Chris Frazar in the Nickerson Lab in Foege at 2:45pm.

He emailed me and Steven (I cc’ed Sam) to get more info on the sample and what kind of sequencing we want. More information on that to come. They will do the library prep and the sequencing.

SamplingApproximately 400 male Tanner crabs, Chionoecetes bairdi, were collected during the Alaska Department of Fish & Game fall survey and transported to the Alaska Fisheries Science Center (AFSC) Auke Bay Laboratories (ABL) in Juneau. Crabs were placed in insulated tanks at approximately 7°C, the temperature of the bottom water where they were collected. Crab biometric data was recorded, and hemolymph samples drawn for preparation of blood smears and preservation in ethanol. Crabs were held at 7°C for 9 days while ethanol-preserved DNA was extracted & Hematodinium sp. infection status determined for each crab with a conventional PCR assay for Hematodinium sp. (Jensen et al. 2010). On Day 9, based on the PCR results, 180 crabs were equally distributed among 9 tanks (10 Hematodinium-positive and 10 Hematodinium-negative crabs per tank) and the surplus crabs were removed from the tanks. Hemolymph samples were taken from the 180 experimental crabs and preserved in RNAlater Stabilization Solution. After the hemolymph draws on Day 9, over the following 2 days, tank temperatures in 3 ‘cold’ tanks and 3 ‘warm’ tanks were adjusted to the experimental treatment temperatures of 4°C and 10°C, respectively, while 3 tanks remained at ‘ambient’ temperature of 7°C; these temperatures were maintained for the duration of the experiment. On Day 11, hemolymph was drawn and preserved in RNAlater. On Day 27, the final hemolymph samples were drawn and preserved in RNAlater and the experiment terminated. RNAlater-preserved samples were transported to the Roberts’ laboratory at the University of Washington for processing.

Figure 1. A, experimental treatment tanks at ABL; B, Grace Crandall, the graduate student on the project, taking a hemolymph sample for preservation in RNAlater from a Tanner crab.

We anticipated high initial mortality of the crabs due to capture and transport stress, reflected by the number of surplus crabs collected, and indeed, lost ~100 crabs before the experiment began. Throughout the experiment, survival in the cold and ambient treatments was higher than anticipated, with 92% of the crabs in each treatment surviving to the end of the experiment. However, in the warm treatment, mortality was high with 50% of the crabs dying between Days 9 and 11, and all but 1 crab per treatment tank succumbing by the end of the experiment.

qPCR assay for Hematodinium infection intensity

In Seattle, DNA was extracted from an aliquot of the Day 27 RNAlater preserved hemolymph. That DNA and DNA extracted from Day 1 ethanol-preserved hemolymph processed in ABL was subjected to a qPCR assay for Hematodinium sp. (Crosson et al., in prep) in order to quantify Hematodinium infection intensity (DNA copy number of Hematodinium). The results will be used to corroborate RNA-Seq analysis results and to investigate the relationship between infection intensity and gene expression levels in the crabs. The qPCR assay, which is more sensitive than the conventional PCR assay, revealed some Hematodinium positive crabs among the crabs that were negative for Hematodinium via the conventional Hematodinium PCR assay. We are using this additional information to inform our sample selection for transcriptome sequencing and analysis.

RNA extraction

Hemolymph samples from 51 crabs (3 sample dates for most crabs, 2 sample dates for some warm treatment crabs: 138 total samples) were processed using a RNA isolation protocol adapted from RNAzol RT. After the completion of the protocol, the isolated RNA was resuspended in 50ul of 0.1% DEPC-treated water, quantified, and stored in -80˚ until sample pooling and/or sequencing. We are currently performing quality assessments on samples for library construction and sequencing that we plan to have underway by the end of this summer.

Based on the timeline in the proposal we are slightly behind schedule in constructing RNA-seq libraries. Due to the high mortality in one treatment group (see below), we decided to evaluate more samples for sequencing than initially planned and to submit samples for sequencing in series, rather than all at the same time. This will allow us to fine tune the sequencing to maximize information from all the crabs, but especially the treatment group that suffered high mortality. We expect to be back on schedule for transcriptome analysis by the end of the year.